Genomic imprinting is the process by which certain genes are marked during gametogenesis to differentially express the parental alleles. The imprints that govern allele-specific expression are established in the germline of the parents during gametogenesis, such that the male parent confers a paternal imprint to his chromosomes during spermatogenesis, and the female parent confers a maternal imprint during oogenesis. This application will focus on the molecular basis for establishment of the paternal imprint during spermatogenesis. Molecular analysis of the Prader-Willi (PWS) and Angelman (AS) syndromes in human 15q11-13 has identified a functional imprinting center (IC) and provided insight into the mechanisms that may regulate imprinting during gametogenesis. Two subregions of the IC within the cluster of imprinted genes in 15q11-13 control the primary paternal- and maternal-specific imprinting of multiple genes. A 3 kb subregion of the IC (the PWS IC) that includes exon 1 and the promoter region of the imprinted SNRPN gene appears to play the primary role in setting the paternal imprint during spermatogenesis. Mutations in this subregion lead to PWS by preventing the establishment of a paternal imprint on genes in 15q11-13 and disrupting imprinting throughout this region in spermatogenesis. Once the primary paternal-specific imprint is set in the PWS IC, this primary imprinting signal must then be transmitted to each imprinted gene in 15q11-13 to establish the paternal pattern of gene expression. Neither the primary imprint nor the mechanism for transmitting the primary IC imprint signal to individual genes are currently known. This application will determine the nature and timing of both the primary and gene-specific imprinting signals in the PWS/AS syntonic region of mouse chromosome 7C during spermatogenesis. Cells from different stages of mouse spermatogenesis will be isolated and examined for differential DNA methylation, DNase I hypersensitive sites, and DNA-protein interactions in mouse 7C that may be indicative of the primary and gene-specific imprinting signals. Understanding the molecular basis of imprinting may lead to therapeutic approaches for treating genetic disorders known to involve imprinting.